Type 1 and Type 2 diabetes mellitus are recognized as bi-hormonal diseases reflecting pathology in both insulin-producing islet b-cells and glucagon-producing islet a-cells. While intensive global efforts have focused on b-cell biology and replacement, little is known about the genetic or molecular effectors of a-cell activity in health or in diabetes, especially in human islets. Discovery of molecular mechanisms that govern human a-cell gene regulation and physiological functions like glucagon output are urgently needed. We have generated new tools to measure human a-cell function and regulation in vivo, and at single-cell resolution. We propose the following scientifically-timely and medically-relevant Aims that will define regulators of human a-cells: 1. Identify mechanisms regulating human a-cell function. To enable unprecedented studies of human glucagon secretion and regulation, we generated NOD.Cg-Prkdc scidIl2rgtm1WjlSz mice (NSG) with an in-frame deletion of preproglucagon exon 3 that encodes Glucagon 1-29; termed GKO-NSG. These mice permit quantification of regulated glucagon secretion from transplanted human a-cells. Genetic, physiological, and genomic approaches in vitro and in vivo will be used to identify mechanisms governing human a-cell function. Specifically, we will analyze how a-cells are impacted by transcription factors, MAFB and RFX6, which our analysis indicates are enriched in a distinct, functionally active cell population. 2. Investigate the role of lipid droplets in human a-cell adaptive responses to lipotoxicity. Lipotoxicity and accompanying insulin resistance is an established risk for diabetes pathogenesis in humans. We will use genetic approaches and physiological assays in vitro and in vivo to identify mechanisms governing accumulation of lipid droplets (LDs), an organelle in human islets. These studies will test the hypothesis that LDs regulate function of human a-cells challenged by lipotoxicity and insulin resistance. This work allows previously unattainable investigation of physiological mechanisms governing and regulating human a-cells. A fundamental advance in diabetes and islet biology research would be the identification and characterization of factors that lead to decreased islet cell function in those with diabetes, or at risk for developing diabetes. Mechanisms governing function of human islet a-cells should be revealed by the studies proposed here. Our work should establish paradigms that connect gene transcriptional and organelle regulation to control of key a-cell physiological functions in healthy and diseased pancreatic islets. Appropriately regulated islet a-cell function is an essential feature of health, and our work will have broad impact by describing ways to ameliorate islet a-cell function, in both normal and pathological conditions.